Pegylated g-csf polypeptides and methods of producing same

ABSTRACT

A method for increasing the stability and uniformity of a PEGylated G-CSF polypeptide having at least one PEG moiety attached to the epsilon amino group of a lysine residue or the N-terminal amino group and at least one PEG moiety attached to a hydroxyl group, comprising subjecting the polypeptide to an elevated pH of above 8.0 for a period of time suitable to remove PEG moieties attached to a hydroxyl group, and reducing the pH to about 8.0 or lower; as well as PEGylated G-CSF polypeptides and compositions produced according to the method and methods for increasing neutrophil levels in a patient using the PEGylated G-CSF polypeptides and compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

Pursuant to 35 U.S.C. §119(e), this application claims the benefit ofU.S. Provisional Application Ser. No. 60/686,726 filed on Jun. 1, 2005,the disclosure of which is incorporated by reference herein in itsentirety for all purposes.

FIELD OF THE INVENTION

The present invention relates to a method for removing labile PEGmoieties from PEGylated G-CSF proteins to increase stability anduniformity, and to the resulting PEGylated G-CSF proteins. The inventionalso relates to pharmaceutical compositions comprising the PEGylatedproteins and methods of treatment by administering the pharmaceuticalcompositions.

BACKGROUND OF THE INVENTION

The covalent attachment of polyethylene glycol (PEG) moieties toproteins or polypeptides (“PEGylation”) is a well-known technique forimproving the properties of such proteins or polypeptides, in particularpharmaceutical proteins, e.g. in order to improve circulation half-lifeand/or to shield potential epitopes and thus reduce the potential for anundesired immunogenic response. Numerous technologies based on activatedPEG are available to provide attachment of the PEG moiety to one or moregroups on the protein. For example, mPEG-succinimidyl propionate(mPEG-SPA, available from Nektar Therapeutics) is generally regarded asbeing selective for attachment to the N-terminus and epsilon-aminogroups of lysine residues via an amide bond. However, in practicemPEG-SPA does not always attach exclusively to these groups, but mayalso attach to the hydroxyl group of a serine, tyrosine or threonineresidue via an ester bond. As a result, PEGylated proteins preparedusing this technology may not have a sufficient degree of uniformity andmay contain a number of different PEG isomers other than those that wereintended. This is undesired for various reasons, including the fact thatit makes characterization of such proteins more complicated. Further,PEG moieties bound to groups other than those intended may be relativelyunstable. For example, PEG moieties bound to a hydroxyl group via anester bond in the case of an amine-specific PEG such as mPEG-SPA tend tobe relatively unstable compared to those moieties that are bound via anamide bond to the N-terminal or a lysine residue. Depending on theformulation and storage conditions, this may lead to an undesired lossof these labile PEG groups and thus potentially to a change in theproperties of the PEGylated protein over time. Further, there may bepotential regulatory or safety issues for a PEGylated proteinpharmaceutical in which there is a risk that one or more of the PEGmoieties may detach from the protein in the body after it isadministered to a patient.

The present invention addresses these problems by providing a method bywhich such labile PEG groups may be easily removed, thereby resulting ina more uniform and stable PEGylated protein product.

BRIEF DISCLOSURE OF THE INVENTION

The present invention relates generally to a method for increasing thestability and uniformity of a PEGylated polypeptide having at least onePEG moiety attached to a lysine residue or the N-terminal and at leastone PEG moiety attached to a hydroxyl group, comprising subjecting thepolypeptide to an altered pH for a period of time suitable to remove PEGmoieties attached to a hydroxyl group, after which the pH is adjusted toa pH suitable for long-term storage of the polypeptide in question.

In a particular aspect, the invention relates to a method for increasingthe stability and uniformity of a PEGylated granulocyte colonystimulating factor (G-CSF) polypeptide having at least one PEG moietyattached to the epsilon amino group of a lysine residue or theN-terminal amino group and at least one PEG moiety attached to ahydroxyl group, comprising subjecting the polypeptide to an elevated pHof above 8.0 for a period of time suitable to remove PEG moietiesattached to a hydroxyl group, and reducing the pH to about 8.0 or lower.

Another aspect of the invention relates to a method for producing aPEGylated G-CSF polypeptide, comprising subjecting a G-CSF polypeptideto a PEGylation reaction with an amine-specific activated polyethyleneglycol (PEG) to produce a PEGylated G-CSF polypeptide intermediate, andsubsequently subjecting the PEGylated polypeptide intermediate to anelevated pH of at least about 9.0 for a period of time suitable toremove PEG moieties attached to a hydroxyl group to produce thePEGylated G-CSG polypeptide.

Another aspect of the invention relates to a composition comprising ahomogeneous mixture of positional PEG isomers of a PEGylated G-CSFpolypeptide variant, wherein at least about 80% of the positional PEGisomers of the mixture consist of two positional PEG isomers each havingPEG moieties consisting of two PEG moieties bound to epsilon aminogroups of lysine and one PEG moiety bound to the N-terminal amino group.

A further aspect of the invention relates to a composition comprising amixture of positional PEG isomers of a PEGylated G-CSF polypeptide,wherein the polypeptide comprises the substitutions K16R, K34R, K40R,T105K and S159K relative to wild-type human G-CSF (SEQ ID NO:1), andwherein at least about 80% of the positional PEG isomers of thepolypeptide are lysine/N-terminal positional PEG isomers having threeattached PEG moieties.

In a still further aspect the invention relates to a method of producinga mixture of lysine/N-terminal positional PEG isomers of a recombinantG-CSF polypeptide comprising the substitutions K16R, K34R, K40R, T105Kand S159K relative to wild-type human G-CSF, comprising: a) expressingthe recombinant G-CSF polypeptide in a host cell; b) isolating therecombinant G-CSF polypeptide; c) reacting the isolated recombinantG-CSF polypeptide with an amine-specific activated PEG to produce aplurality of positional PEG isomers of the recombinant G-CSFpolypeptide; d) reacting the plurality of positional PEG isomers at a pHof 8.5 to 10.5 to produce a plurality of partially dePEGylatedlysine/N-terminal positional PEG isomers of the recombinant G-CSFpolypeptide.

Further aspects of the invention relate to PEGylated G-CSF polypeptidesproduced using the methods described herein, as well as PEGylated G-CSFpolypeptides characterized by their positional PEG isomer distributionand compositions comprising such PEGylated G-CSF polypeptides, andmethods for increasing the level of neutrophils in a patient sufferingfrom or at risk of an insufficient neutrophil level by administering aPEGylated G-CSF polypeptide or a composition of the invention.Additional aspects of the invention and preferred embodiments will beapparent from the description below as well as from the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a scan of an SDS-PAGE analysis of a G-CSF variant that wasPEGylated, partially de-PEGylated, and purified as described herein.

FIG. 2 shows a scan of an SDS-PAGE analysis of a G-CSF that wasPEGylated and partially de-PEGylated as described herein.

FIG. 3 shows a cation exchange chromatogram of a partially de-PEGylatedproduct pool prepared according to the invention.

FIG. 4 shows a cation exchange chromatogram of a purified, PEGylatedG-CSF variant that was not subject to de-PEGylation according to theinvention.

DESCRIPTION Definitions

In the context of the present description and claims the followingdefinitions apply:

The terms “polypeptide” or “protein” may be used interchangeably hereinto refer to polymers of amino acids, without being limited to an aminoacid sequence of any particular length. These terms are intended toinclude not only full-length proteins but also e.g. fragments ortruncated versions, variants, domains, etc. of any given protein orpolypeptide.

A “G-CSF” polypeptide is a polypeptide having the sequence of humangranulocyte 10 colony stimulating factor (hG-CSF) as shown in SEQ IDNO:1, or a variant of hG-CSF that exhibits G-CSF activity. The “G-CSFactivity” may be the ability to bind to a G-CSF receptor (Fukunaga etal., J. Bio. Chem., 265:14008, 1990), but is preferably G-CSF cellproliferation activity, in particular determined in an in vitro activityassay using the murine cell line NFS-60 (ATCC Number: CRL-1838). Asuitable in vitro assay for G-CSF activity using the NFS-60 cell line isdescribed in by Hammerling et al. in J. Pharm. Biomed. Anal. 13(1):9-20,1995. A polypeptide “exhibiting” G-CSF activity is considered to havesuch activity when it displays a measurable function, e.g. a measurableproliferative activity in the in vitro assay.

A “variant” is a polypeptide which differs in one or more amino acidresidues from a parent polypeptide, where the parent polypeptide isgenerally one with a native, wild-type amino acid sequence, typically anative mammalian polypeptide, and more typically a native humanpolypeptide. The variant thus contains one or more substitutions,insertions or deletions compared to the native polypeptide. These may,for example, include truncation of the N- and/or C-terminus by one ormore amino acid residues, or addition of one or more extra residues atthe N- and/or C-terminus, e.g. addition of a methionine residue at theN-terminus. The variant will most often differ in up to 15 amino acidresidues from the parent polypeptide, such as in up to 12, 10, 8 or 6amino acid residues.

An “amine-specific activated PEG” is any activated PEG derivative thatpreferentially attaches to the N-terminal amino group or the ε-aminogroups of lysine residues via an amide bond. Examples of amine-specificactivated PEG derivatives include:

mPEG-succinimidyl propionate (mPEG-SPA):

mPEG-succinimidyl butanoate (mPEG-SBA):

and mPEG-succinimidyl α-methylbutanoate (mPEG-SMB):

mPEG-SPA, mPEG-SBA and mPEG-SMB are available from Nektar Therapeutics;see the Nektar Advanced PEGylation Catalogs 2004 and 2005-2006,“Polyethylene Glycol and Derivatives for Advanced PEGylation”. Otheramine-specific activated PEG derivatives include PEG-SS (SuccinimidylSuccinate), PEG-SG (Succinimidyl Glutarate), PEG-NPC (p-nitrophenylcarbonate), and PEG-isocyanate, available from SunBio Corporation; andPEG-SCM, available from NOF Corporation.

A “labile” PEG moiety refers in the present context to a PEG moietyattached to a hydroxyl group of a polypeptide, in particular via anester bond to the hydroxyl group of a serine, tyrosine or threonineresidue. As explained above, the attachment of such PEG moieties via ahydroxyl group tends to be unstable, so that these PEG moieties tend todetach from the polypeptide over time through hydrolysis of the esterbonds, e.g. when a polypeptide containing such moieties is stored in theform of an aqueous solution.

As used herein, “stability” refers to the stability of the attachment ofPEG moieties bound to a polypeptide, i.e. whether such PEG moietiesremain bound to the polypeptide over time, e.g. during storage in anaqueous solution, or whether they tend to detach e.g. as a result ofester bond hydrolysis.

As used herein the term “positional PEG isomer” of a protein refers todifferent PEGylated forms of the protein where PEG groups are located atdifferent amino acid positions of the protein. The term“lysine/N-terminal PEG isomer” of a protein means that the PEG groupsare attached to the amino-terminal of the protein and/or to epsilonamino groups of lysine residues in the protein. For example, the phrase“lysine/N-terminal positional PEG isomers having 3 attached PEGmoieties”, as applied to G-CSF, means a mixture of G-CSF positional PEGisomers in which three PEG groups are attached to epsilon amino groupsof lysine residues and/or to the N-terminus of the protein. Typically, a“lysine/N-terminal positional PEG isomer having 3 attached PEG moieties”will have two PEG moieties attached to lysine residues and one PEGmoiety attached to the N-terminus. Analysis of the positional PEGisomers may be performed using cation exchange HPLC as described in theexamples below.

A “substantially purified mixture of lysine/N-terminal positional PEGisomers” of a polypeptide refers to mixture of lysine/N-terminalpositional PEG isomers which has been subjected to a chromatographic orother purification procedure in order to remove impurities such asnon-lysine/N-terminal positional PEG isomers. The “substantiallypurified mixture of lysine/N-terminal positional PEG isomers” will, forexample, be free of most labile PEG moieties attached to a hydroxylgroup that would otherwise be present in the absence of a partialde-PEGylation step and subsequent purification as described herein, andwill typically contain less than about 20% polypeptides containing alabile PEG moiety attached to a hydroxyl group, more typically less thanabout 15%. Preferably, there will be less than about 10% polypeptidescontaining a labile PEG moiety attached to a hydroxyl group, for exampleless than about 5%.

The term “homogeneous mixture of positional PEG isomers of a polypeptidevariant” means that the polypeptide moiety of the different positionalPEG isomers is the same. This means that the different positional PEGisomers of the mixture are all based on a single polypeptide variantsequence. For example, a homogeneous mixture of positional PEG isomersof a PEGylated G-CSF polypeptide variant means that different positionalPEG isomers of the mixture are based on a single G-CSF polypeptidevariant.

As used herein, “uniformity” refers to the homogeneity of a PEGylatedpolypeptide in terms of the number of different positional isomers, i.e.different polypeptide isomers containing different numbers of PEGmoieties attached at different positions, as well as the relativedistribution of these positional isomers. For pharmaceuticalpolypeptides intended for therapeutic use in humans or animals, it isgenerally desirable that the number of different positional PEG isomersis minimized.

“Partial de-PEGylation” refers to the fact that the methods of theinvention serve to remove labile PEG moieties attached to a hydroxylgroup, while PEG moieties that are more stably attached to theN-terminal or the amino group of a lysine residue remain intact. Asexplained below, the progress of the de-PEGylation process is dependentupon the particular elevated pH value and the duration at the elevatedpH. Therefore, depending on the conditions chosen in any particularcase, it is possible that a small proportion of the polypeptides canstill have a PEG moiety attached to a hydroxyl group at the end of ade-PEGylation step. Based on the information provided herein and commongeneral knowledge in the art of protein analysis, however, personsskilled in the art will be able to adapt the process so thatsubstantially all labile PEG moieties are removed and any remaininglabile PEG moieties are insignificant for the desired properties of thefinal product. Any reference to “de-PEGylation” herein should beunderstood to refer to “partial de-PEGylation”.

DETAILED DESCRIPTION

As indicated above, one particular aspect of the invention relates to amethod for increasing the stability and uniformity of a PEGylated G-CSFpolypeptide having at least one PEG moiety attached to the epsilon aminogroup of a lysine residue or the N-terminal amino group and at least onePEG moiety attached to a hydroxyl group, comprising subjecting thepolypeptide to an elevated pH of above 8.0 for a period of time suitableto remove PEG moieties attached to a hydroxyl group, and reducing the pHto about 8.0 or lower. Although this method is suitable for anymammalian G-CSF polypeptide, the polypeptide is preferably a human G-CSFpolypeptide, i.e. with the amino acid sequence of SEQ ID NO:1,optionally with a methionine residue added at the N-terminal, or avariant thereof. In the case of G-CSF variants, these may have one ormore substitutions, insertions or deletions, and/or truncation of the N-and/or C-terminus by one or more amino acid residues, compared to anative, wild-type G-CSF polypeptide, typically compared to human G-CSF(SEQ ID NO:1), for example from 1 to 15 substitutions, such as up to 6,8, 10 or 12 substitutions. Preferred G-CSF variants include thosedisclosed in WO 01/51510 and WO 03/006501.

As it is described e.g. in WO 03/006501, hG-CSF contains four lysineresidues in positions 16, 23, 34 and 40, and when subjecting G-CSF toPEGylation using activated PEG that preferentially binds to lysineresidues or the N-terminal it will often be desirable to remove at leastone of these lysine residues, e.g. two, three or all of these residues,e.g. by deletion but preferably by substitution. The G-CSF polypeptideused in the method of the invention may therefore be a variant in whichat least one of the amino acid residues selected from the groupconsisting of K16, K23, K34 and K40 has been deleted or substituted withanother amino acid residue. Typically, removal of a lysine residue willbe by substitution, generally by substitution with an R or Q residue,preferably an R residue. The G-CSF polypeptide may thus have one, two,three or four substitutions selected from the group consisting ofK16R/Q, K23R/Q, K34R/Q, K40R/Q (where, for example, “K16R/Q” denotesthat the lysine in position 16 is substituted with either an arginine ora glutamine residue). Preferably, the polypeptide includes thesubstitutions K16R+K34R+K40R, while the lysine in position 23 is leftunaltered.

Along with removal of lysine residues where PEGylation is not desired,WO 03/006501 also describes addition of lysine residues in order tocreate sites for PEGylation. These may be added by insertion but arepreferably added by substitution. Examples of preferred amino acidsubstitutions in order to introduce a site for PEGylation include one ormore of Q70K, Q90K, T105K, Q120K, T133K, S159K and H170K, such as two,three, four or five of these substitutions, with preferred substitutionsbeing T105K+S159K. In a preferred embodiment the G-CSF polypeptideincludes the substitutions K16R, K34R, K40R, T105K and S159K. In aparticular embodiment, the G-CSF polypeptide has only these fivesubstitutions relative to hG-CSF.

Production of G-CSF Polypeptides

The G-CSF polypeptides to be PEGylated in accordance with the inventionmay be produced by any suitable method known in the art, for example asdescribed in WO 03/006501, which is hereby incorporated by reference.Such methods include constructing a nucleotide sequence encoding thepolypeptide and expressing the sequence in a suitable transformed ortransfected host. However, polypeptides of the invention may beproduced, albeit less efficiently, by chemical synthesis or acombination of chemical synthesis or a combination of chemical synthesisand recombinant DNA technology. A nucleotide sequence encoding apolypeptide or the polypeptide part of a conjugate of the invention maybe constructed by isolating or synthesizing a nucleotide sequenceencoding the parent G-CSF, such as hG-CSF with the amino acid sequenceshown in SEQ ID NO:1, and then changing the nucleotide sequence so as toeffect introduction (i.e. insertion or substitution) or deletion (i.e.removal or substitution) of the relevant amino acid residue(s).

The nucleotide sequence is conveniently modified by site-directedmutagenesis in accordance with conventional methods. Alternatively, thenucleotide sequence is prepared by chemical synthesis, e.g. by using anoligonucleotide synthesizer, wherein oligonucleotides are designed basedon the amino acid sequence of the desired polypeptide, and preferablyselecting those codons that are favored in the host cell in which therecombinant polypeptide will be produced. For example, several smalloligonucleotides coding for portions of the desired polypeptide may besynthesized and assembled by PCR, ligation or ligation chain reaction(LCR) (Barany, PNAS 88:189-193, 1991). The individual oligonucleotidestypically contain 5′ or 3′ overhangs for complementary assembly.

Once assembled (by synthesis, site-directed mutagenesis or anothermethod), the nucleotide sequence encoding the polypeptide is insertedinto a recombinant vector and operably linked to control sequencesnecessary for expression of the G-CSF in the desired transformed hostcell.

Persons skilled in the art will be capable of selecting suitablevectors, expression control sequences and hosts for expressing thepolypeptide. The recombinant vector may be an autonomously replicatingvector, i.e. a vector, which exists as an extrachromosomal entity, thereplication of which is independent of chromosomal replication, e.g. aplasmid. Alternatively, the vector is one which, when introduced into ahost cell, is integrated into the host cell genome and replicatedtogether with the chromosome(s) into which it has been integrated.

The vector is preferably an expression vector in which the nucleotidesequence encoding the polypeptide of the invention is operably linked toadditional segments required for transcription of the nucleotidesequence. The vector is typically derived from plasmid or viral DNA. Anumber of suitable expression vectors for expression in the host cellsmentioned herein are commercially available or described in theliterature. Detailed information on suitable vectors for expressingG-CSF may be found in WO 03/006501.

The term “control sequences” is defined herein to include all componentswhich are necessary or advantageous for the expression of thepolypeptide of the invention. Each control sequence may be native orforeign to the nucleic acid sequence encoding the polypeptide. Suchcontrol sequences include, but are not limited to, a leader sequence,polyadenylation sequence, propeptide sequence, promoter, enhancer orupstream activating sequence, signal peptide sequence, synthetic intron,and transcription terminator. At a minimum, the control sequencesinclude a promoter. A wide variety of expression control sequences maybe used in the present invention, e.g. any of the control sequencesdisclosed in WO 03/006501.

The nucleotide sequence of the invention encoding a polypeptideexhibiting G-CSF activity, whether prepared by site-directedmutagenesis, synthesis, PCR or other methods, may optionally alsoinclude a nucleotide sequence that encodes a signal peptide. The signalpeptide is present when the polypeptide is to be secreted from the cellsin which it is expressed. Such signal peptide, if present, should be onerecognized by the cell chosen for expression of the polypeptide. Thesignal peptide may be homologous (e.g. be that normally associated withhG-CSF) or heterologous (i.e. originating from another source thanhG-CSF) to the polypeptide or may be homologous or heterologous to thehost cell, i.e. be a signal peptide normally expressed from the hostcell or one which is not normally expressed from the host cell.Accordingly, the signal peptide may be prokaryotic, e.g. derived from abacterium such as E. coli, or eukaryotic, e.g. derived from a mammalian,or insect or yeast cell. For further information on suitable signalpeptides, see WO 03/006501.

Any suitable host may be used to produce the G-CSF polypeptide,including bacteria (although not particularly preferred), fungi(including yeasts), plant, insect, mammal, or other appropriate animalcells or cell lines, as well as transgenic animals or plants. Mammaliancells are preferred. Examples of bacterial host cells includegram-positive bacteria such as strains of Bacillus, e.g. B. brevis or B.subtilis, Pseudomonas or Streptomyces, or gram-negative bacteria, suchas strains of E. coli. Examples of suitable filamentous fungal hostcells include strains of Aspergillus, e.g. A. oryzae, A. niger, or A.nidulans, Fusarium or Trichoderma. Examples of suitable yeast host cellsinclude strains of Saccharomyces, e.g. S. cerevisiae,Schizosaccharomyces, Klyveromyces, Pichia, such as P. pastoris or P.methanolica, Hansenula, such as H. Polymorpha or Yarrowia. Examples ofsuitable insect host cells include a Lepidoptora cell line, such asSpodoptera frugiperda (Sf9 or Sf21) or Trichoplusioa ni cells (HighFive) (U.S. Pat. No. 5,077,214). Examples of suitable mammalian hostcells include Chinese hamster ovary (CHO) cell lines, (e.g. CHO-K1; ATCCCCL-61), Green Monkey cell lines (COS) (e.g. COS1 (ATCC CRL-1650), COS 7(ATCC CRL-1651)); mouse cells (e.g. NS/O), Baby Hamster Kidney (BHK)cell lines (e.g. ATCC CRL-1632 or ATCC CCL-10), and human cells (e.g.HEK 293 (ATCC CRL-1573)). Additional suitable cell lines are known inthe art and available from public depositories such as the American TypeCulture Collection, Rockville, Md.

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods known in the art. For example, the cell may becultivated by shake flask cultivation, small-scale or large-scalefermentation (including continuous, batch, fed-batch, or solid statefermentations) in laboratory or industrial fermenters performed in asuitable medium and under conditions allowing the polypeptide to beexpressed and/or isolated. The cultivation takes place in a suitablenutrient medium comprising carbon and nitrogen sources and inorganicsalts, using procedures known in the art. Suitable media are availablefrom commercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The resulting polypeptide may be recovered by methods known in the art.For example, the polypeptide may be recovered from the nutrient mediumby conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray drying, evaporation, orprecipitation.

The polypeptides may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Jansonand Lars Ryden, editors, VCH Publishers, New York, 1989). Specificmethods for purifying polypeptides exhibiting G-CSF activity aredescribed by D. Metcalf and N. A. Nicola in The hemopoieticcolony-stimulating factors, p. 50-51, Cambridge University Press (1995),by C. S. Bae et al., Appl. Microbiol. Biotechnol, 52:338-344 (1999) andin U.S. Pat. No. 4,810,643.

PEGylation

The purified G-CSF polypeptides can then be PEGylated by a variety ofmethods known to one of ordinary skill in the art. For example, a G-CSFpolypeptide can be subjected to PEGylation with an amine-specificactivated polyethylene glycol (PEG).

The amine-specific activated PEG may be any of those mentioned above,for example mPEG-succinimidyl propionate (mPEG-SPA), mPEG-succinimidylbutanoate (mPEG-SBA), or mPEG-succinimidyl α-methylbutanoate (mPEG-SMB).The molecular weight of the PEG moieties may be selected based e.g. onthe number of PEG moieties to be attached to the polypeptide and willoften be in the range of from about 1 kDa to about 20 kDa, such as fromabout 1 kDa to about 12 kDa, e.g. from about 2 kDa to about 10 kDa, suchas from about 4 kDa to about 6 kDa, e.g. about 5 kDa. For example, theactivated PEG may be mPEG-SPA with a molecular weight of about 5 kDa.PEGylation may, for example, be performed at a pH of about 7.5-9.0, e.g.about 8.0-8.5. When used about PEG moieties herein, the word “about”indicates an approximate average molecular weight and reflects the factthat there will normally be a certain molecular weight distribution in agiven polymer preparation. For further general information on PEGylationmethods see, for example, the Nektar Advanced PEGylation Catalogs 2004and 2005-2006, as well as the references cited therein. A more detaileddescription of PEGylation methods for G-CSF polypeptides is alsoprovided in WO 03/006501.

Partial de-PEGylation

The choice of an elevated pH for the de-PEGylation step is made based onfactors such as the temperature at which the de-PEGylation step takesplace, the duration of the de-PEGylation step, and the conditions thatwere used for PEGylation, including the pH at which PEGylation wasperformed. Generally, the higher the pH used during de-PEGylation, thefaster the de-PEGylation takes place. Persons skilled in the art willthus realize that de-PEGylation conditions such as pH, time andtemperature can be adjusted to each other to obtain the desired resultin any given situation. For example, although it is possible to use arelatively low elevated pH of e.g. between 8 and 9, use of a higher pHof e.g. between 9 and 11, such as between 9 and 10, will result in afaster de-PEGylation. Typically, the elevated pH will therefore be inthe range of from about 8.5 to about 11.0, e.g. from about 8.5 to about10.5, such as from about 9.0 to about 10.0. The elevated pH may, forexample, be in the range of from about 9.2 to about 9.8, e.g. about 9.5.

Normally, the de-PEGylation step will take place immediately followingPEGylation, as this will generally be most efficient and result inshorter processing times and higher yields. In its basic version, theinvention is thus characterized by partial de-PEGylation of a PEGylatedbulk intermediate, followed by conventional separation using e.g.chromatographic purification as described below. If desired for anyreason, however, the PEGylation procedure may be followed by anintermediate purification step prior to de-PEGylation. The intermediatepurification step may, e.g., comprise chromatographic purification asdescribed below followed by ultrafiltration and/or diafiltration. Theintermediate product obtained from any such intermediate purificationprocedure may, if desired, be stored prior to de-PEGylation and anysubsequent purification steps. In this case, the intermediate PEGylatedproduct may e.g. be stored by freezing or lyophilization using methodsgenerally known in the art.

The de-PEGylation may be performed at any temperature which is otherwisesuitable for the polypeptide in question, for example from about 5° C.to about 40° C., such as from about 10° C. to about 35° C. Thede-PEGylation will often be performed at a temperature of from about 15°C. to about 25° C., e.g. at an ambient temperature of about 20-22° C.

As explained above, the duration of the de-PEGylation step at anelevated pH to obtain a desired result will depend on other factors suchas pH and temperature, but will generally be in the range of from about2 hours to about 100 hours, typically from about 4 hours to about 72hours, more typically from about 8 hours to about 48 hours. Since itwill normally be desirable to perform the de-PEGylation step in as shorta time as possible, the polypeptide will often be subjected to theelevated pH for a period of not more than about 24 hours, e.g. fromabout 12 hours to about 30 hours, such as from about 18 to about 24hours. As indicated above, a relatively short de-PEGylation period atthe elevated pH will generally be accompanied by a relatively high pH,e.g. about 9-10.

Following the de-PEGylation at an elevated pH, the pH is reduced toabout 8.0 or lower, the particular pH being chosen according to theintended storage conditions for the polypeptide. For G-CSF, a suitablepH for long-term storage will often be in the range of from about 2.0 toabout 5.0, for example from about 2.5 to about 4.5, such as about 4.0.In some cases, depending e.g. on the particular formulation to be used,a somewhat higher pH may be chosen, i.e. a pH of about 5.0-8.0, e.g.about 6.0-7.5, such as about 7.0-7.5. Various pharmaceuticalformulations of G-CSF are described, e.g., in U.S. Pat. No. 5,104,651,U.S. Pat. No. 5,919,757, U.S. Pat. No. 6,497,869 and U.S. Pat. No.6,776,983.

For adjustment of pH in the context of the present invention, anysuitable acid or base may be used, both organic or inorganic acids andbases. Similarly, any suitable buffer may be used for maintaining the pHat a desired level. Persons skilled in the art will be familiar withacids, bases and buffers suitable for use in any given situation,depending e.g. on the polypeptide in question and the desired pH.

Subsequent to reduction of the pH, the polypeptide will generally besubjected to at least one chromatographic purification step, for exampleion exchange chromatography or gel filtration chromatography, in orderto separate polypeptides having a desired number of PEG groups attached.The chromatographic purification may be carried out using methodsgenerally known in the art. For example, where the reduced pH followingde-PEGylation is below about 7.0, cation exchange chromatography may beused. Alternatively or additionally, chromatography may be performedprior to reduction of the pH, using anion exchange chromatography at theelevated pH value. Further purification as well as any desired analysisof the product may similarly be performed using standard methods knownin the art. For example, further purification subsequent to thechromatographic purification step may be performed using e.g.ultrafiltration or diafiltration.

As mentioned above, another aspect of the invention relates to a methodfor producing a PEGylated G-CSF polypeptide, comprising subjecting aG-CSF polypeptide to PEGylation with an amine-specific activatedpolyethylene glycol (PEG), and subsequently subjecting the PEGylatedpolypeptide to an elevated pH of at least about 9.0 for a period of timesuitable to remove PEG moieties attached to a hydroxyl group. Thismethod results in a PEGylated G-CSF polypeptide having at least one PEGmoiety attached to the epsilon amino group of a lysine residue or theN-terminal amino group and substantially no PEG moieties attached to ahydroxyl group. In one embodiment, this aspect of the invention isperformed using PEGylation at a pH of about 7.0-9.0, after which the pHis increased to remove labile PEG moieties. In another embodiment,PEGylation may be performed at a higher pH of above 9.0, such as a pH inthe range of 9-11, such as from about 9.2 to 10.0, e.g. about 9.5. Inthis case, PEGylation and de-PEGylation may be performed as a singlestep at a single pH value for a sufficient period of time to result inthe desired PEGylation at the N-terminus and lysine residues whileavoiding undesired binding of PEG moieties to hydroxyl groups.

The amine-specific activated PEG may be any of those mentioned above,for example mPEG-succinimidyl propionate (mPEG-SPA), mPEG-succinimidylbutanoate (mPEG-SBA), or mPEG-succinimidyl α-methylbutanoate (mPEG-SMB).The molecular weight of the PEG moieties will similarly be as describedabove, e.g. in the range of from about 1 kDa to about 20 kDa, such asfrom about 1 kDa to about 12 kDa, e.g. from about 2 kDa to about 10 kDa,such as from about 4 kDa to about 6 kDa, e.g. about 5 kDa. For example,the activated PEG may be mPEG-SPA with a molecular weight of about 5kDa. PEGylation may, for example, be performed at a pH of about 7.5-9.0,e.g. about 8.0-8.5. Depending on the pH used during PEGylation, theelevated pH chosen will also be similar to the ranges generallydescribed above, for example in the range of from about 9.2 to 11.0,such as from about 9.2 to 10.0. Similarly, the time period at theelevated pH may also be chosen as described above, and the same appliesto subsequent purification, etc.

The method described herein provides a number of advantages forproduction processes aimed at producing a uniform and stable PEGylatedprotein, including the fact that it is simple and fast, requiring only asingle extra step of altered pH for a limited time period, and there isno need to otherwise change the PEGylation or purification proceduresused in a given production process. For this reason, the same generalprocedure of an altered pH for a period of time sufficient to removelabile PEG groups may be used for the production of any PEGylatedprotein. In general, either a basic or acidic pH may be used forremoving labile PEG moieties attached to a hydroxyl group, with thedetachment of the labile PEG moieties taking place faster when the pH ismore acidic (for an acid pH) or more basic (for a basic pH). The choiceof conditions for obtaining labile PEG detachment will be made based one.g. the structural features of the particular protein, including themicroenvironment of attachment sites for PEG moieties.

PEGylated G-CSF Polypeptides

As indicated above, the method of the present invention is advantageousin that it results in an increased homogeneity of the PEGylatedpolypeptide in terms of the number of different positional PEG isomers.Depending on the number of attachment groups and the particularPEGylation chemistry being used, the method allows the isolation ofPEGylated G-CSF that is more uniform and more stable during storage thanwould otherwise be possible. For example, it has been found that for aPEGylated G-CSF polypeptide having the substitutions K16R, K34R, K40R,T105K and S159K relative to wild-type human G-CSF, it is possible toobtain a product wherein at least about 90-95% of the positional PEGisomers of the polypeptide have 3 attached PEG moieties.

Another embodiment of the invention relates to a composition comprisinga homogeneous mixture of positional PEG isomers of a PEGylated G-CSFpolypeptide variant, wherein at least about 80%, preferably at leastabout 85%, more preferably at least about 90%, for example at leastabout 95% of the positional PEG isomers of the mixture consist of twopositional PEG isomers each having PEG moieties consisting of two PEGmoieties bound to epsilon amino groups of lysine residues and one PEGmoiety bound to the N-terminal amino group.

Another embodiment of the invention thus relates to a PEGylated G-CSFpolypeptide comprising the substitutions K16R, K34R, K40R, T105K andS159K relative to wild-type human G-CSF, wherein at least about 80% ofthe positional PEG isomers of the polypeptide have 3 attached PEGmoieties. Typically, the proportion of the positional PEG isomers having3 attached PEG moieties will be greater than 80%, such as at least about85% or at least about 90%, and may be even higher, such as at leastabout 91%, at least about 92%, at least about 93%, at least about 94%,or at least about 95%. In yet another embodiment, the invention relatesto a PEGylated G-CSF polypeptide containing only the substitutions K16R,K34R, K40R, T105K and S159K relative to wild-type human G-CSF, andwherein at least about 80% of the positional PEG isomers of thepolypeptide have 3 attached PEG moieties. Typically, the proportion ofthe positional PEG isomers having 3 attached PEG moieties will begreater than 80%, such as at least about 85% or at least about 90%, andmay be even higher, such as at least about 91%, at least about 92%, atleast about 93%, at least about 94%, or at least about 95%.

In a still further embodiment, the invention relates to a compositioncomprising a mixture of PEGylated G-CSF polypeptides comprising thesubstitutions K16R, K34R, K40R, T105K and S159K relative to wild typehuman G-CSF, wherein at least about 80%, preferably at least about 85%,more preferably at least about 90%, such as at least about 95% of thePEGylated G-CSF polypeptides in the mixture are composed of twopositional PEG G-CSF isomers each containing three PEG groups, whereinone of the isomers has PEG groups attached at the N-terminal, Lys23 andLys159 and the other isomer has PEG groups attached at the N-terminal,Lys105 and Lys159. In yet another embodiment, the invention relates to acomposition comprising a mixture of PEGylated G-CSF polypeptidescontaining only the substitutions K16R, K34R, K40R, T105K and S159Krelative to wild type human G-CSF, and wherein at least about 80%,preferably at least about 85%, more preferably at least about 90%, suchas at least about 95% of the PEGylated G-CSF polypeptides in the mixtureare composed of two positional PEG G-CSF isomers each containing threePEG groups, wherein one of the isomers has PEG groups attached at theN-terminal, Lys23 and Lys159 and the other isomer has PEG groupsattached at the N-terminal, Lys105 and Lys159

It has been found that PEGylated G-CSF polypeptides produced accordingto the invention are highly storage-stable with respect to their degreeof PEGylation. As it is shown in the examples below, aqueousformulations of such PEGylated G-CSF polypeptides are able to be storedfor an extended period of time with a largely unaltered PEGylationpattern. This was in particular the case when the compositions werestored at a temperature of 5° C., but surprisingly the compositions werefound to be relatively stable even when stored at a high temperature of25° C. or 35° C.

A further aspect of the invention therefore relates to a storage-stablePEGylated G-CSF polypeptide comprising the substitutions K16R, K34R,K40R, T105K and S159K relative to wild-type human G-CSF, wherein atleast about 80% of the positional PEG isomers of the polypeptide have 3attached PEG moieties after storage in an aqueous reference compositionfor 3 months at a temperature of 5° C. Typically, the proportion of thepositional PEG isomers having 3 attached PEG moieties after storage for3 months at a temperature of 5° C. will be greater than 80%, such as atleast about 85% or at least about 90%. In some cases, this proportionmay even higher, such as at least about 91%, at least about 92%, atleast about 93%, at least about 94%, or at least about 95%. For purposesof determining the stability of a PEGylated G-CSF polypeptide producedaccording to the invention, the stability may be tested as described inExample 4, for example using one of formulations A, B, C or D describedtherein as the reference composition, in particular formulation D.

Pharmaceutical Compositions

Another aspect of the invention relates to pharmaceutical compositionscomprising a PEGylated G-CSF polypeptide according to the invention. Oneembodiment of the invention thus relates to a composition comprising apharmaceutically acceptable carrier and a PEGylated G-CSF polypeptide,wherein the polypeptide comprises the substitutions K16R, K34R, K40R,T105K and S159K relative to wild-type human G-CSF (SEQ ID NO:1), andwherein at least about 80% of the positional PEG isomers of thepolypeptide have 3 attached PEG moieties. Typically, the proportion ofthe positional PEG isomers having 3 attached PEG moieties will be atleast about 85%, preferably at least about 90%, and may be even higher,such as at least about 91%, at least about 92%, at least about 93%, atleast about 94%, or at least about 95%. In yet another embodiment, theinvention relates to a pharmaceutical composition comprising apharmaceutically acceptable carrier and a PEGylated G-CSF polypeptide,wherein the polypeptide contains only the substitutions K16R, K34R,K40R, T105K and S159K relative to wild-type human G-CSF (SEQ ID NO:1),and wherein at least about 80% of the positional PEG isomers of thepolypeptide have 3 attached PEG moieties. Typically, the proportion ofthe positional PEG isomers having 3 attached PEG moieties will be atleast about 85%, preferably at least about 90%, and may be even higher,such as at least about 91%, at least about 92%, at least about 93%, atleast about 94%, or at least about 95%.

Another embodiment of the invention relates to a pharmaceuticalcomposition comprising a homogeneous mixture of positional PEG isomersof a PEGylated G-CSF polypeptide variant, wherein at least about 80%,preferably at least about 85%, more preferably at least about 90%, forexample at least about 95% of the positional PEG isomers of the mixtureconsist of two positional PEG isomers each having PEG moietiesconsisting of two PEG moieties bound to epsilon amino groups of lysineresidues and one PEG moiety bound to the N-terminal amino group.

In a further embodiment the invention relates to pharmaceuticalcompositions comprising a pharmaceutically acceptable carrier and amixture of PEGylated G-CSF polypeptides comprising the substitutionsK16R, K34R, K40R, T105K and S159K relative to wild type human G-CSF,wherein at least about 80%, preferably at least about 85%, morepreferably about 90%, such as at least about 95% of the PEGylated G-CSFpolypeptides in the mixture are composed of two positional PEG G-CSFisomers each containing three PEG groups, wherein one of the isomers hasPEG groups attached at the N-terminal, Lys23 and Lys159 and the otherisomer has PEG groups attached at the N-terminal, Lys105 and Lys159. Inyet another embodiment, the invention, the invention relates topharmaceutical compositions comprising a pharmaceutically acceptablecarrier and a mixture of PEGylated G-CSF polypeptides containing onlythe substitutions K16R, K34R, K40R, T105K and S159K relative to wildtype human G-CSF, wherein at least about 80%, preferably at least about85%, more preferably about 90%, such as at least about 95% of thePEGylated G-CSF polypeptides in the mixture are composed of twopositional PEG G-CSF isomers each containing three PEG groups, whereinone of the isomers has PEG groups attached at the N-terminal, Lys23 andLys159 and the other isomer has PEG groups attached at the N-terminal,Lys105 and Lys159

Pharmaceutical compositions comprising the G-CSF polypeptide of theinvention may be formulated in a variety of forms, e.g. in lyophilisedform or, preferably, in a stable liquid formulation, typically as anaqueous formulation. Such compositions typically comprise one or morecomponents selected from buffering agents, preservatives,isotonicifiers, stabilizers, non-ionic surfactants or detergents, andadditional miscellaneous excipients such as bulking agents or fillers,chelating agents, antioxidants and cosolvents. One example of a suitableformulation for G-CSF is that used for Neulasta® (pegfilgrastim), whichis supplied in an aqueous solution with a pH of 4.0 containing, per 0.6ml, 0.35 mg acetate, 30.0 mg sorbitol, 0.02 mg polysorbate 20, and 0.02mg sodium.

An example of a pharmaceutical composition is a solution designed forparenteral administration. Although in many cases pharmaceuticalsolution formulations are provided in liquid form, appropriate forimmediate use, such parenteral formulations may also be provided infrozen or in lyophilized form. In the former case, the composition mustbe thawed prior to use. The latter form is often used to enhance thestability of the active compound contained in the composition under awider variety of storage conditions, as it is recognized by thoseskilled in the art that lyophilized preparations are generally morestable than their liquid counterparts. Such lyophilized preparations arereconstituted prior to use by the addition of one or more suitablepharmaceutically acceptable diluents such as sterile water for injectionor sterile physiological saline solution. The compositions of theinvention are preferably in the form of an aqueous solution, however.

In case of parenterals, they are prepared for storage as lyophilizedformulations or aqueous solutions by mixing, as appropriate, thepolypeptide having the desired degree of purity with one or morepharmaceutically acceptable carriers, excipients or stabilizerstypically employed in the art (all of which may be termed “excipients”),for example buffering agents, stabilizing agents, preservatives,isotonifiers, non-ionic detergents, antioxidants and/or othermiscellaneous additives.

Buffering agents help to maintain the pH in a desired range. They aretypically present at a concentration ranging from about 2 mM to about 50mM. Suitable buffering agents include both organic and inorganic acidsand salts thereof such as citrate buffers (e.g., monosodiumcitrate-disodium citrate mixture, citric acid-trisodium citrate mixture,citric acid-monosodium citrate mixture, etc.), succinate buffers (e.g.,succinic acid-monosodium succinate mixture, succinic acid-sodiumhydroxide mixture, succinic acid-disodium succinate mixture, etc.),tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaricacid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture,etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture,fumaric acid-disodium fumarate mixture, monosodium fumarate-disodiumfumarate mixture, etc.), gluconate buffers (e.g., gluconic acid-sodiumglyconate mixture, gluconic acid-sodium hydroxide mixture, gluconicacid-potassium glyuconate mixture, etc.), oxalate buffers (e.g., oxalicacid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture,oxalic acid-potassium oxalate mixture, etc.), lactate buffers (e.g.,lactic acid-sodium lactate mixture, lactic acid-sodium hydroxidemixture, lactic acid-potassium lactate mixture, etc.) and acetatebuffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodiumhydroxide mixture, etc.). Additional possibilities are phosphatebuffers, histidine buffers and trimethylamine salts such as Tris.

Preservatives are added to retard microbial growth, and when present aretypically added in amounts of about 0.2%-1% (w/v). Suitablepreservatives include phenol, benzyl alcohol, meta-cresol, methylparaben, propyl paraben, octadecyldimethylbenzyl ammonium chloride,benzalkonium halides (e.g. benzalkonium chloride, bromide or iodide),hexamethonium chloride, alkyl parabens such as methyl or propyl paraben,catechol, resorcinol, cyclohexanol and 3-pentanol.

Isotonicifiers are added to ensure isotonicity of liquid compositionsand include polyhydric sugar alcohols, preferably trihydric or highersugar alcohols, such as glycerin, erythritol, arabitol, xylitol,sorbitol and mannitol. Polyhydric alcohols can be present in an amountbetween 0.1% and 25% by weight, typically 1% to 5%, taking into accountthe relative amounts of the other ingredients.

Stabilizers refer to a broad category of excipients which can range infunction from a bulking agent to an additive which solubilizes thetherapeutic agent or helps to prevent denaturation or adherence to thecontainer wall. Typical stabilizers can be polyhydric sugar alcohols(enumerated above); amino acids such as arginine, lysine, glycine,glutamine, asparagine, histidine, alanine, ornithine, L-leucine,2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugaralcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol,xylitol, ribitol, myoinisitol, galactitol, glycerol and the like,including cyclitols such as inositol; polyethylene glycol; amino acidpolymers; sulfur-containing reducing agents, such as urea, glutathione,thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglyceroland sodium thiosulfate; low molecular weight polypeptides (i.e. <10residues); proteins such as human serum albumin, bovine serum albumin,gelatin or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructoseand glucose; disaccharides such as lactose, maltose and sucrose;trisaccharides such as raffinose, and polysaccharides such as dextran.Stabilizers may e.g. be present in the range of from 0.1 to 10,000 partsby weight based on the active protein weight.

Non-ionic surfactants or detergents (also known as “wetting agents”) maybe present to help solubilize the therapeutic agent as well as toprotect the therapeutic polypeptide against agitation-inducedaggregation, which also permits the formulation to be exposed to shearsurface stress without causing denaturation of the polypeptide. Suitablenon-ionic surfactants include polysorbates (20, 80, etc.), polyoxamers(184, 188 etc.), Pluronic® polyols, polyoxyethylene sorbitan monoethers(Tween®-20, Tween®-80, etc.).

Additional miscellaneous excipients that may be added include bulkingagents or fillers (e.g. starch), chelating agents (e.g. EDTA),antioxidants (e.g., ascorbic acid, methionine, vitamin E) andcosolvents.

The active ingredient may also be entrapped in microcapsules prepared,for example, by coascervation techniques or by interfacialpolymerization, for example hydroxymethylcellulose, gelatin orpoly-(methylmethacylate) microcapsules, in colloidal drug deliverysystems (for example liposomes, albumin microspheres, microemulsions,nano-particles and nanocapsules) or in macroemulsions.

Parenteral formulations to be used for in vivo administration must besterile. This is readily accomplished by methods well-known in the art,for example, by filtration through sterile filtration membranes.

Therapeutic Methods

Another aspect of the invention relates to therapeutic methods andmethods for the manufacture of a medicament using the PEGylated G-CSFpolypeptide of the invention. Leukopenia (a reduced level of white bloodcells) and neutropenia (a reduced level of neutrophils) are disordersthat result in an increased susceptibility to various types ofinfections. Neutropenia can be chronic, e.g. in patients infected withHIV, or acute, e.g. in cancer patients undergoing chemotherapy orradiation therapy. For patients with severe neutropenia, e.g. as aresult of chemotherapy, even relatively minor infections can be serious,and neutropenia will often require an interruption in the chemotherapyprotocol. The PEGylated G-CSF polypeptides of the invention areparticularly suitable for prevention or treatment of infection in cancerpatients undergoing certain types of chemotherapy, radiation therapy andbone marrow transplantations, mobilisation of progenitor cells forcollection in peripheral blood progenitor cell transplantations,treatment of severe chronic or relative leukopenia, treatment ofpatients with acute myeloid leukemia, treatment of AIDS or otherimmunodeficiency diseases, and for antifungal therapy, in particular fortreatment of systemic or invasive candidiasis. A “patient” for thepurposes of the present invention includes both humans and othermammals, although the therapeutic methods of the invention are primarilyaimed at treatment of human patients.

This aspect of the invention thus relates to a method for increasing thelevel of neutrophils in a patient suffering from or at risk of aninsufficient neutrophil level, comprising administering to said patientan effective dose of a PEGylated G-CSF polypeptide as described above,or a pharmaceutical composition as described above comprising thePEGylated G-CSF polypeptide. In one embodiment of this aspect of theinvention, the patient suffering from or at risk of an insufficientneutrophil level is a cancer patient, in particular a cancer patientbeing treated with chemotherapy or radiation therapy, especiallychemotherapy.

The PEGylated G-CSF polypeptides of the invention are contemplated to beuseful for stimulating production of neutrophils in patients sufferingfrom a variety of different types of cancer, including breast cancer,non-small cell lung cancer, small cell lung cancer, colorectal cancer,uterine cancer, ovarian cancer, non-Hodgkin's lymphoma (NHL) andHodgkin's disease. Similarly, the polypeptides of the invention arecontemplated for administration to patients receiving any of a varietyof different types of chemotherapeutic agents, including:

-   -   alkylating agents, e.g. mustard gas derivatives, such as        Cyclophosphamide, Chlorambucil, Ifosfamide, Mechlorethamine or        Melphalan; ethylenimines, such as Hexamethylmelamine or        Thiotepa; alkylsulfonates such as Busulfan; hydrazines and        triazines such as Altretamine, Dacarbazine, Procarbazine or        Temozolomide; nitrosureas such as Carmustine, Lomustine or        Streptozocin; and inorganic metal complex agents such as        Cisplatin, Carboplatin or Oxaliplatin;    -   plant alkaloids, e.g. taxanes (e.g., Docetaxel or Paclitaxel),        vinca alkaloids (e.g., Vinblastine, Vincristine or Vinorelbine),        camptothecan analogs (e.g., Irinotecan or Topotecan) and        podophyllotoxins (e.g., Etoposide or Tenisopide);    -   antitumor antibiotics, e.g. anthracyclines, such as        Daunorubicin, Doxorubicin, Epirubicin, Idarubicin, or        Mitoxantrone; chromomycins, such as Dactinomycin or Plicamycin;        and other antitumor antibiotics such as Bleomycin or Mitomycin;    -   antimetabolites, e.g. folic acid antagonists, such as        Methotrexate; pyrimidine antagonists, such as Capecitabine,        Cytarabine, 5-Fluorouracil (5-FU), Foxuridine or Gemcitabine;        purine antagonists, such as 6-Mercaptopurine or 6-Thioguanine;        adenosine deaminase inhibitors, such as Cladribine, Fludarabine,        Nelarabine or Pentostatin; and ribonucleotide reductase        inhibitors, such as Hydroxyurea;    -   topoisomerase inhibitors, e.g. topoisomerase I inhibitors, such        as Ironotecan or Topotecan; and topoisomerase II inhibitors,        such as Amsacrine, Etoposide, Etoposide Phosphate or Teniposide.

The PEGylated G-CSF polypeptides of the invention will be administeredto patients in an “effective” or “therapeutically effective” dose, i.e.a dose that is sufficient to produced the desired effects in relation tothe condition for which it is administered, in particular a dose thatunder the given conditions is sufficient to result in the desiredstimulation of neutrophils. The exact dose may depend on factors such asthe individual patient and the disorder being treated, and will be ableto be determined by one skilled in the art, typically a medical doctor.The dosage of the PEGylated G-CSF polypeptide may e.g. be approximatelythe same order of magnitude as the current recommended dosage forpegfilgrastim (Neulasta®), which is 6 mg per adult patient. Anappropriate dose of the PEGylated G-CSF polypeptide of the invention istherefore contemplated to be in the range of from about 1 mg to about 15mg, such as from about 2 mg to about 15 mg, e.g. from about 3 mg toabout 12 mg. A suitable dose may thus be, for example, about 3 mg, about6 mg, or about 9 mg. In each case, the dosages are expressed as astandard dose per patient, where the patient is an adult or otherwiseweighs at least 45 kg. Alternatively, the dosage may be determinedaccording to the weight of the patient, such that an appropriate dose ofthe G-CSF polypeptide of the invention is in the range of from about 10μg/kg to about 200 μg/kg, such as from about 25 μg/kg to about 150μg/kg, e.g. from about 30 μg/kg to about 120 μg/kg. A suitable dose maythus be, for example, about 30 μg/kg, about 60 μg/kg, about 90 μg/kg orabout 120 μg/kg.

The invention is further described by the following non-limitingexamples.

EXAMPLES Example 1 Preparation and Analysis of a Partially de-PEGylatedG-CSF Variant

Sample Preparation and de-PEGylation

A G-CSF variant having the substitutions K16R, K34R, K40R, T105K andS159K compared to wild-type human G-CSF (SEQ ID NO:1) was produced fromCHO-K1 cells substantially as described in WO 03/006501. 200 mL of 4.5mg/mL of the G-CSF variant (900 mg G-CSF) was PEGylated using mPEG-SPA5000 (Nektar Therapeutics). Briefly, 100 mL of a 13.2% (w/w) solution ofmPEG-SPA 5000 was added over a period of 10 minutes to the 200 mL ofG-CSF solution and gently stirred to ensure sufficient mixing. Thesample was allowed to incubate for 44 minutes at 21±3° C., pH 8.5, withgentle stirring. The sample mixture was subsequently adjusted to pH 9.5using a stock solution of 800 mM boric acid pH 10.0. The sample was thenincubated at 21±3° C. for 24 hours without stirring. The sample was thendiluted approx. 2.5 fold with 100 mmol/kg citric acid, 20 mmol/kg NaOH,pH 2.5, to a final pH of 3.5.

Isolation of Partially de-PEGylated G-CSF

After reduction of the pH to 3.5, the sample was loaded immediately ontoa VL44 (Millipore) column packed with approx. 225 mL of SP-Sepharose HP(Amersham, GE Healthcare) equilibrated with 20 mmol/kg citric acid, 15mmol/kg NaOH, pH 3.4. After loading of the material, the column waswashed to remove unbound material such as free mPEG-acid. A lineargradient of 25-100% elution buffer (20 mmol/kg citric acid, 20 mmol/kgNaOH, 200 mmol/kg NaCl, pH 3.5) was used to elute the PEGylated G-CSFfrom the column. The gradient was developed for 25 CV (column volumes).The column step was performed at 21±3° C. and all buffers used wereadjusted to the same temperature. The column load was 4.1 mg protein/mlresin and the flow was 8 CV/hour.

Fractions having a sample size of 40 mL were collected and analysed bySDS-PAGE. Based on the SDS-PAGE analysis a product pool of 1440 mL,corresponding to about 6.5 CV, containing isolated G-CSF havingprimarily 3 attached PEG groups per G-CSF molecule was made. It iscontemplated that the volume of the product pool can be reduced by 10increasing the steepness of the elution gradient.

The product pool was diafiltered to approx. 200 mL and subsequentlydiafiltered into 10 mM sodium acetate, 43 mg/mL, pH 4.0 and concentratedto 4.9 mg/mL using a Millipore LabScale TFF-system equipped with two 50cm² Millipore Pellicon XL membranes with a molecular weight cutoff(MWCO) of 30 kDa.

Results

The product pool was ultrafiltered and diafiltered into 10 mMNa-acetate, 43 mg/ml mannitol, pH 4.0 and subjected to physico-chemicalcharacterization. FIG. 1 shows a scan of the SDS-PAGE analysis of theG-CSF variant that was PEGylated, partially de-PEGylated, and purifiedas described above, the seven lanes shown being as follows:

1 Molecular weight marker 2 After PEGylation 3 pH 9.5, T = 0 h 4 pH 9.5,T = 24 h 5 Upon application on cation exchange column 6 Flow throughfrom cation exchange 7 Purified product

A single major band in lane 7 is observed, corresponding to the G-CSFvariant carrying 3 mPEG-SPA 5000 groups per polypeptide molecule. Inaddition, a minor band corresponding to G-CSF carrying 4 mPEG-SPA 5000groups is also seen in lane 7. If desired, optimization or adjustment ofthe purification procedure by methods generally known in the art willallow this minor band corresponding to 4 PEG groups to be reduced orsubstantially eliminated, although this will lead to a slight reductionof the yield depending on the degree of purity required.

The yield of this procedure was 450 mg of purified, partiallyde-PEGylated G-CSF carrying primarily 3 PEG groups per polypeptidemolecule, corresponding to an overall yield of 50%.

Example 2 Preparation and Analysis of a Partially de-PEGylated G-CSFVariant

Sample Preparation, de-PEGylation and Purification

A G-CSF variant having the substitutions K16R, K34R, K40R, T105K andS159K 10 compared to wild-type human G-CSF (SEQ ID NO:1) was producedfrom CHO-K1 cells and subsequently PEGylated using mPEG-SPA 5000substantially as described in Example 1 above, resulting in 29 mL of 3.5mg/mL PEGylated G-CSF variant. This was diafiltrated into 150 mM sodiumborate, pH 9.5, using a Vivaspin filter device equipped with a 10 kDaMWCO filter. The final sample concentration was 4.7 mg/mL. The samplewas then incubated at 21±3° C. for 42 hours.

After incubation, the sample was prepared for cation exchangechromatography by dilution with 30 mM citric acid, 10 mM NaOH, pH 2.9.The sample was then applied onto an XK 16/20 column (AmershamBiosciences) packed with 28 mL of SP-Sepharose HP resin. The column wasequilibrated with an equilibration buffer of 20 mM citric acid, 15 mMNaCl, pH 3.5, prior to sample loading. After loading, the column waswashed with the same buffer and eluted using a linear gradient of from15 mM to 200 mM NaCl in 20 mM citric acid, pH 3.5, to separate thedifferent PEGylated species.

Fractions were collected in tubes and analysed by SDS-PAGE analysis. Aproduct pool containing the PEGylated G-CSF variant primarily containing3 attached PEG moieties was made based on the SDS-PAGE. This productpool was diafiltrated and buffer exchanged using Vivaspin filter deviceswith a MWCO of 10 kDa. The sample was diafiltrated into 10 mM sodiumsuccinate, 43 mg/mL, pH 4.0, and finally concentrated to 3.0 mg/mL.

Positional Isomer Analysis

Using cation exchange HPLC (CIEX-HPLC) combined with a sialidasepretreatment data was obtained on the composition of differentpositional PEG isomers in product pools containing multiple PEGylatedspecies. In addition to heterogeneity after PEGylation due to thepresence of different positional isomers, PEGylated G-CSF is alsoheterogeneous in that it contains one O-glycosylation site (Thr133) thatcan have zero, one or two sialic acid groups attached. In order toreduce the number of peaks in the chromatogram as a result of differentnumbers of sialic acid groups, a sialidase enzyme pre-treatment step isincluded prior to analysis. The sialidase treatment is performed byfirst diluting the G-CSF solution, if necessary, to 1 mg/ml with a 50 mMsodium acetate buffer, pH 5.0. after which 0.05 μl (0.25 mUnit) ofsialidase is added per μg protein, and the sample is incubated at 37° C.for 18 hours to remove the sialic acids. The sample is then analysed thesame day or kept at 4° C. until the day of analysis. HPLC was performedusing a PolySULFOETHYL A™ cation exchange HPLC column (PolyLC Inc.),with UV detection at 214 nm. Characterization of the different peaks wasperformed by SDS-PAGE analysis after manual collection from thePolySULFOETHYL A™ CIEX column.

Results—PEG Loss

The buffer-exchanged sample was analysed by SDS-PAGE to estimate thedegree of PEG loss as a function of time. The results (see FIG. 2) weresimilar to those illustrated in FIG. 1 for Example 1, with only minimalchanges in the overall degree of PEGylation being observed betweensamples incubated for 24 or 42 hours (FIG. 2, lanes 4 and 5).

Results—Assessment of Positional Isomers in Product Pools

FIG. 3 shows a cation exchange chromatogram of the partiallyde-PEGylated product pool according to the invention obtained asdescribed above containing primarily three PEG 5000 groups as judged bySDS-PAGE analysis. The two major peaks each represent one majorpositional isomer carrying three PEG 5000 groups. The three minor peaksrepresent two positional isomers carrying four PEG groups and onepositional isomer carrying two PEG groups.

The two major peaks were collected and subjected to native peptide mapanalysis to determine the position of the attached PEG groups. It wasfound that the two major positional isomers are PEGylated on thefollowing amino acid residues:

Isomer A: N-terminal, Lys23 and Lys159

Isomer B: N-terminal, Lys105 and Lys159

These two major isomers represented more than 95% of the positionalisomers present in the sample.

Example 3 Comparative Example

For comparative purposes, FIG. 4 shows a cation exchange chromatogram ofa purified, PEGylated G-CSF variant that was not subject tode-PEGylation according to the present invention. The G-CSF variant inthis case had the same five substitutions compared to native human G-CSFas indicated above in Examples 1 and 2, i.e. K16R, K34R, K40R, T105K andS159K. It was prepared and PEGylated with mPEG-SPA 5000 substantially asdescribed in Example 1, with the exception of the fact that it was notsubjected to incubation at pH 9.5 for removal of labile PEG moieties. Asa result, the PEGylated variant comprised a mixture of a number ofdifferent PEG isomers having 2-6 attached PEG moieties. This mixture ofpositional isomers was purified using cation exchange chromatographysubstantially as described in Example 1. A fraction containing primarily4-5 attached PEG moieties was isolated and, after ultrafiltration anddiafiltration substantially as described in Example 1, was subjected toCIEX-HPLC analysis as described above. This fraction corresponds to the3-PEG fraction described in Examples 1 and 2, but with additional(labile) PEG groups being attached at one or two positions relative tothe partially de-PEGylated 3-PEG product, i.e. the purified product inthis case primarily contains 4-5 attached PEG groups.

Compared to the partially de-PEGylated product prepared according to theinvention whose composition is shown in FIG. 3, the product shown inFIG. 4 is clearly a much more complex and heterogeneous mixture. Whereasthe product prepared according to the invention shown in FIG. 3 containsmore than 95% of the two major positional isomers each having 3 PEGmoieties, the product shown in FIG. 4 comprises six major positionalisomers each having either 4 or 5 attached PEG moieties. These six 4-5PEG positional isomers comprise labile PEG moieties at one or both ofSer66 or Tyr165 (data not shown), as well as stable PEG moieties at theN-terminal and at 1 or 2 of positions K23, K105 and K159. Due to sterichindrance, not all of these positions on a single G-CSF polypeptide canbe occupied by PEG moieties. The product shown in FIG. 4 thus contains aheterogeneous mixture of the six following major PEG isomers:

N-terminal, K23, S66, K159, Y165 (5-PEG)

N-terminal, S66, K105, K159, Y165 (5-PEG)

N-terminal, K23, S66, K159 (4-PEG)

N-terminal, K23, S66, Y165 (4-PEG)

N-terminal, S66, K105, K159 (4-PEG)

N-terminal, S66, K105, Y165 (4-PEG)

Example 4 Stability Data

The stability of the attached PEG moieties was tested in differentaqueous formulations stored at four different temperatures. Thepolypeptide was a G-CSF variant having the five substitutions listed inExample 1 and which was prepared, PEGylated, and subject to partialde-PEGylation and then purified also as described in Example 1. Theformulations tested were as follows:

Formulation Units A B C D G-CSF mg/ml 2 10 2 2 Na-Acetate mM 10 10 10 10Tween ®80 mg/ml — — 0.05 — Tween ®20 mg/ml — — — 0.05 Mannitol mg/ml 4343 43 43The pH of each of the formulations was 4.0.

Using CIEX-HPLC, the distribution of PEG isomers in each sample wasdetermined after 26 days, 56 days and 89 days to investigate thestability of the PEGylation. The results are shown in the table below.

Others/not Formulation Temp. Day 3 PEG characterized 2 PEG A — 0 92.37.8 0 −80° C. 26 93.3 6.6 0 5° C. 93.5 6.4 0.1 25° C. 94.1 5.5 0.3 35°C. 94.3 4.7 0.8 −80° C. 56 95.4 4.7 0 5° C. 95.3 4.7 0 25° C. 95.3 3.80.9 35° C. 95.3 3.0 1.7 −80° C. 89 96.1 3.9 0 5° C. 95.9 4.1 0 25° C.96.1 3.1 0.8 35° C. 95.5 2.7 1.8 B — 0 92.4 7.6 0 −80° C. 26 93.5 6.50.0 5° C. 93.4 6.5 0.1 25° C. 94.0 5.7 0.3 35° C. 94.2 5.0 0.8 −80° C.56 95.1 4.9 0 5° C. 95.4 4.7 0 25° C. 95.2 4.3 0.6 35° C. 96.2 2.4 1.4−80° C. 89 95.9 4.1 0 5° C. 95.9 4.1 0 25° C. 96.8 3.2 0 35° C. 95.5 2.61.9 C — 0 92.1 7.9 0 −80° C. 26 93.8 6.1 0 5° C. 94.1 5.8 0 25° C. 94.05.7 0.3 35° C. 94.6 4.5 0.8 −80° C. 56 95.1 4.9 0 5° C. 95.1 4.9 0 25°C. 94.1 4.8 1.1 35° C. 95.1 3.0 1.9 −80° C. 89 95.9 4.1 0 5° C. 95.9 4.10 25° C. 96.1 3.2 0.7 35° C. 95.5 2.7 1.8 D — 0 92.6 7.5 0 −80° C. 2693.8 6.1 0.1 5° C. 93.6 6.3 0.1 25° C. 93.7 6.1 0.3 35° C. 94.4 4.9 0.8−80° C. 56 95.3 4.7 0 5° C. 95.2 4.8 0 25° C. 95.2 4.0 0.8 35° C. 95.53.3 1.3 −80° C. 89 96.0 4.0 0 5° C. 96.0 4.0 0 25° C. 96.2 3.1 0.7 35°C. 95.7 2.5 1.8

The above results show that regardless of the formulation and thestorage temperature, the PEGylation is highly stable, with theproportion of 3 PEG isoforms being very close to 95% at all temperatureswhen sampled at either 26, 56 or 89 days. The only difference readilyseen from the table is a slight decrease over time, using the −80° C.data as a reference, in the proportion of the other/non-characterizedforms, mainly for samples stored at 25° C. or 35° C., and acorresponding increase in the proportion of 2 PEG isomers for thesesamples.

By comparison, the table below shows similar data, determined after 20and 83 days of storage, for the same G-CSF variant in a formulationsimilar to those described above, but prepared without the de-PEGylationstep of the present invention, i.e. corresponding to the productdescribed in comparative Example 3 above. The formulation in this casehad a pH of 4.0 and consisted of 15 mM sodium acetate, 45 mg/mlmannitol, and 5 mg/ml of the PEGylated G-CSF variant, which at thebeginning of the experiment contained primarily (98-99%) 4-5 attachedPEG moieties. The table below shows the distribution of the PEG isoformsas determined after 83 days at the four temperatures indicated as wellas after 20 days at two of these temperatures. It is seen that theproportion of the G-CSF polypeptides containing 4-5 PEG moietiesdecreases just slightly after 83 days when stored at 5° C., while thereis a substantial loss of 4-5 PEG isoforms and a corresponding increasein 3 PEG isoforms when stored at 25° C., and a far greater loss of 4-5PEG isoforms and corresponding increase in 3 PEG isoforms at 35° C. Thisillustrates that in the absence of the de-PEGylation method of thepresent invention, a PEGylated G-CSF polypeptide prepared as describedabove contains labile PEG groups that over time and depending on thetemperature will be detached from the polypeptide when stored in anaqueous formulation.

Others/not Temp. Day 4-5 PEG 3 PEG characterized — 0 98.6% 0 1.4% −80°C.  20 98.1% 0.5% 1.4% 35° C. 96.0% 2.9% 1.1% −80° C.  83 97.9% 0.6%1.6%  5° C. 97.6% 1.0% 1.5% 25° C. 90.6% 8.4% 1.1% 35° C. 78.1% 21.5%0.5%

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. It is understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and scope of the appended claims. All publications, patents,patent applications, and/or other documents cited in this applicationare incorporated herein by reference in their entirety for all purposesto the same extent as if each individual publication, patent, patentapplication, and/or other document were individually indicated to beincorporated herein by reference in its entirety for all purposes.

1.-39. (canceled)
 40. A method for increasing the level of neutrophilsin a patient suffering from or at risk of an insufficient neutrophillevel, comprising administering to said patient an effective dose of aPEGylated G-CSF polypeptide produced by a method comprising: subjectinga G-CSF polypeptide to a PEGylation reaction with an amine-specificactivated polyethylene glycol (PEG) to produce a PEGylated G-CSFpolypeptide intermediate having at least one PEG moiety attached to theepsilon amino group of a lysine residue or the N-terminal amino groupand at least one PEG moiety attached to a hydroxyl group, and subjectingthe PEGylated G-CSF polypeptide intermediate to an elevated pH of above8.0 for a period of time suitable to remove PEG moieties attached to ahydroxyl group, and reducing the pH to a reduced pH of about 8.0 orlower, thereby producing the PEGylated G-CSF polypeptide.
 41. The methodof claim 40, wherein the insufficient neutrophil level is a result ofchemotherapy or radiation therapy. 42.-47. (canceled)
 48. A method forincreasing the level of neutrophils in a patient suffering from or atrisk of an insufficient neutrophil level, comprising administering tosaid patient an effective dose of a composition comprising a homogeneousmixture of positional PEG isomers of a PEGylated G-CSF polypeptidevariant, wherein at least about 80% of the positional PEG isomers of themixture consist of two positional PEG isomers each having PEG moietiesconsisting of two PEG moieties bound to epsilon amino groups of lysineand one PEG moiety bound to the N-terminal amino group.
 49. (canceled)50. The method of claim 40, wherein the amine-specific activated PEG isselected from the group consisting of mPEG-succinimidyl propionate(mPEG-SPA), mPEG-succinimidyl butanoate (mPEG-SBA), or mPEG-succinimidylα-methylbutanoate (mPEG-SMB).
 51. The method of claim 50, wherein theamine-specific activated PEG is mPEG-SPA with a molecular weight ofabout 5 kDa.
 52. The method of claim 40, wherein the G-CSF polypeptideis a G-CSF variant comprising the substitutions K16R, K34R, K40R, T105Kand S159K relative to wild-type human G-CSF.
 53. A method for increasingthe level of neutrophils in a patient suffering from or at risk of aninsufficient neutrophil level, comprising administering to said patientan effective dose of a composition comprising a mixture of positionalPEG isomers of a PEGylated G-CSF polypeptide, wherein the G-CSFpolypeptide comprises the substitutions K16R, K34R, K40R, T105K andS159K relative to wild-type human G-CSF, and wherein at least about 80%of the positional PEG isomers of the polypeptide are lysine/N-terminalpositional PEG isomers having three attached PEG moieties.
 54. Themethod of claim 53, wherein the insufficient neutrophil level is aresult of chemotherapy or radiation therapy.
 55. The method of claim 48,wherein the insufficient neutrophil level is a result of chemotherapy orradiation therapy.